Molecular Phylogeny of the Chipmunks Inferred from Mitochondrial Cytochrome b and Cytochrome Oxidase II Gene Sequences

There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used to further test the fit of models of evolution to the cyt b data. Other sciurid cyt b sequence was added to examine the evolution of Tamias in the context of other sciurids. Relationships among Tamias species are discussed, particularly the possibility of a current sorting event among taxa of the southwestern United States and the extreme divergences among the three subgenera (Neotamias, Eutamias, and Tamias).     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

Coevolutionary Patterns in Cytochrome c Oxidase Subunit I Depend on Structural and Functional Context

The strength and pattern of coevolution between amino acid residues vary depending on their structural and functional environment. This context dependence, along with differences in analytical technique, is responsible for the different results among coevolutionary analyses of different proteins. It is thus important to perform detailed study of individual proteins to gain better insight into how context dependence can affect coevolutionary patterns even within individual proteins, and to unravel the details of context dependence with respect to structure and function. Here we extend our previous study by presenting further analysis of residue coevolution in cytochrome c oxidase subunit I sequences from 231 vertebrates using a statistically robust phylogeny-based maximum likelihood ratio method. As in previous studies, a strong overall coevolutionary signal was detected, and coevolution within structural regions was significantly related to the C_α distances between residues. While the strong selection for adjacent residues among predicted coevolving pairs in the surface region indicates that the statistical method is highly selective for biologically relevant interactions, the coevolutionary signal was strongest in the transmembrane region, although the distances between coevolving residues were greater. This indicates that coevolution may act to maintain more global structural and functional constraints in the transmembrane region. In the transmembrane region, sites that coevolved according to polarity and hydrophobicity rather than volume had a greater tendency to colocalize with just one of the predicted proton channels (channel H). Thus, the details of coevolution in cytochrome c oxidase subunit I depend greatly on domain structure and residue physicochemical characteristics, but proximity to function appears to play a critical role. We hypothesize that coevolution is indicative of a more important functional role for this channel. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Characterization of Taenia multiceps Isolates from Gansu Province,China by Sequencing of Mitochondrial Cytochrome C Oxidase Subunit 1

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.     

Molecular Phylogeny of the Chipmunk Genus Tamias Based on the Mitochondrial Cytochrome Oxidase Subunit II Gene

Complete sequences of the mitochondrial cytochrome oxidase subunit II gene were used to construct a phylogeny for 21 of the 25 currently recognized chipmunk species. Phylogenetic analyses indicate that T. striatus (subgenus Tamias, eastern United States) and T. sibiricus (subgenus Eutamias, Asia) are distantly related to the other species (subgenus Neotamias), which constitute a western North American radiation. We discuss and compare our molecular phylogeny to previous taxonomies and present a suggested classification of the species groups for the subgenus Neotamias.     

淮河水系日本沼虾群体遗传结构和系统演化的线粒体COI序列分析

测定了淮河水系17个日本沼虾(Macrobrachium nipponense)野生群体共248个个体的线粒体细胞色素氧化酶亚基I(COI)部分序列,获得623 bp核苷酸片段,包括48个变异位点,定义了31个单倍型,共享单倍型有12个,整体单倍型多样性和平均核苷酸多样性均处于中间水平。AMOVA分析表明,17个群体间的遗传分化系数Fst=0.041 3(P0.05),群体间遗传分化较小。Kimura 2-paramter遗传距离在五河与焦岗湖、花家湖及瓦埠湖群体间最大,为0.014,在高邮和邵伯湖群体之间最小,为0.003。MP系统树与单倍型进化网络关系图具有较高的一致性,31个单倍型被分为3个进化枝,其中一个进化枝主要以下游群体为主,另外2个进化枝主要以中游群体为主。群体中性检验、错配分析表明,淮河日本沼虾近期曾经历过种群扩张。     

Evolution of the primate cytochrome c oxidase subunit II gene

We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. Correspondence to: R.L. Honeycutt     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

A Candidate Complex Approach to Study Functional Mitochondrial DNA Changes: Sequence Variation and Quaternary Structure Modeling of Drosophila simulans Cytochrome c Oxidase

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for a Conformational Change in Subunit III of Bovine Heart Mitochondrial Cytochrome c Oxidase1

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl—biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.     

Structural Study of the Interaction Between the Mitochondrial Presequence of Cytochrome c Oxidase Subunit IV and Model Membranes

The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of -helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.     

Molecular phylogeography of two sibling species of <Emphasis Type="Italic">Eurema</Emphasis> butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.     

Evolution of the Mitochondrial Cytochrome Oxidase II Gene in Collembola

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data. Received: 12 January 1996 / Accepted: 10 August 1996     

Phylogeny and host-specificity of European seed beetles (Coleoptera, Bruchidae), new insights from molecular and ecological data

We used partial sequences of three mitochondrial genes (12S rRNA, cytochrome b, and cytochrome c oxidase subunit I) to reconstruct the phylogeny of European seed beetles (Bruchidae) belonging to the genera Bruchus Linnaeus and Bruchidius Schilsky. Adult beetles examined in this study were obtained from larvae bred from seeds directly collected in the field. Parsimony, maximum likelihood, and Bayesian inference were used to infer phylogenetic relationships among species. Both genera, Bruchidius and Bruchus, formed monophyletic groups in all analyses. Our results were partially in discrepancy with existing taxonomic groups (Borowiec, 1987). Critical analysis of relationships among taxa, and exhaustive review of host-plants data highlight the very high level of specialization of these seed beetles. Phylogenetically related insects were associated with host-plants belonging to the same botanical tribes.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.     

Molecular systematics of Acarus siro s. lat., a complex of stored food pests

The astigmatid mite Acarus siro (Linnaeus 1758) is an important agricultural pest and environmental allergen. However, it is likely that many mites described in the literature as A. siro, collected from both outdoor and stored product habitats, may belong to one of its sibling species, A. farris [Ent. Ber. Amst. 2 (26) (1905) 20] or A. immobilis [Bull. Br. Mus. Nat. Hist. 11 (1964a) 413; Acarologia. 6 (Suppl) (1964) 101]. The three species are difficult to separate morphologically, gene exchange between some of them is possible and, although each species displays environmental preferences, they occur together in some environments. This raises a question about their separate species status. In a pilot study, we investigated whether genetic data supported the separate species status of these forms. Both nuclear (the second internal transcribed spacer region [ITS-2] of the ribosomal cistron) and mitochondrial (cytochrome oxidase subunit I, mtcoxI hereafter) loci were employed for this purpose. Mtcox1 data does not conflict the differentiation into three separate species and while the ITS2 data were problematic for this group of mites it suggested that a congener, Acarus gracilis [Ann. Mag. Nat. Hist. 10 (1957) 753], is basal to the A. siro species complex.     

Molecular Evolution of a Small Gene Family of Wound Inducible Kunitz Trypsin Inhibitors in Populus

Maximum likelihood models of codon substitutions were used to analyze the molecular evolution of a Kunitz trypsin inhibitor (KTI) gene family in Populus and Salix. The methods support previous assertions that the KTI genes comprise a rapidly evolving gene family. Models that allow for codon specific estimates of the ratio of nonsynonymous to synonymous substitutions (ω) among sites detect positive Darwinian selection at several sites in the KTI protein. In addition, branch-specific maximum likelihood models show that there is significant heterogeneity in ω among branches of the KTI phylogeny. In particular, ω is substantially higher following duplication than speciation. There is also evidence for significant rate heterogeneity following gene duplication, suggesting different evolutionary rates in newly arisen gene duplicates. The results indicate uneven evolutionary rates both between sites in the KTI protein and among different lineages in the KTI phylogeny, which is incompatible with a neutral model of sequence evolution. [Reviewing Editor: Dr. Willie J. Swanson]     

A Theoretical Analysis of the Redox-Coupled Protonation of Heme a and CuA in the Cytochrome c Oxidase with CO-Inhibited Binuclear Center

One of the proposed mechanisms of functioning of cytochrome c oxidase (COX) postulates that hemea is the element pumping protons across the membrane. It is generally believed that, to support this mechanism, a substantial proton uptake/release should exist upon heme a reduction/oxidation. Two direct measurements of proton uptake/release in oxidation/reduction of heme a in CO-bound mixed-valence COX were recently reported. In this paper, we develop a general formalism for the interpretation of such experiments and discuss the results of these experiments. A control experiment is proposed to verify the conclusions made in previous studies.